Toward the improvement of total nitrogen deposition budgets in the United States.

Frameworks for limiting ecosystem exposure to excess nutrients and acidity require accurate and complete deposition budgets of reactive nitrogen (Nr). While much progress has been made in developing total Nr deposition budgets for the U.S., current budgets remain limited by key data and knowledge gaps. Analysis of National Atmospheric Deposition Program Total Deposition (NADP/TDep) data illustrates several aspects of current Nr deposition that motivate additional research. Averaged across the continental U.S., dry deposition contributes slightly more (55%) to total deposition than wet deposition and is the dominant process (>90%) over broad areas of the Southwest and other arid regions of the West. Lack of dry deposition measurements imposes a reliance on models, resulting in a much higher degree of uncertainty relative to wet deposition which is routinely measured. As nitrogen oxide (NOx) emissions continue to decline, reduced forms of inorganic nitrogen (NHx = NH3 + NH4+) now contribute >50% of total Nr deposition over large areas of the U.S. Expanded monitoring and additional process-level research are needed to better understand NHx deposition, its contribution to total Nr deposition budgets, and the processes by which reduced N deposits to ecosystems. Urban and suburban areas are hotspots where routine monitoring of oxidized and reduced Nr deposition is needed. Finally, deposition budgets have incomplete information about the speciation of atmospheric nitrogen; monitoring networks do not capture important forms of Nr such as organic nitrogen. Building on these themes, we detail the state of the science of Nr deposition budgets in the U.S. and highlight research priorities to improve deposition budgets in terms of monitoring and flux measurements, leaf- to regional-scale modeling, source apportionment, and characterization of deposition trends and patterns.

Nitrogen isotopes as indicators of NO(x) source contributions to atmospheric nitrate deposition across the midwestern and northeastern United States.

Global inputs of NO(x) are dominated by fossil fuel combustion from both stationary and vehicular sources and far exceed natural NO(x) sources. However, elucidating NO(x) sources to any given location remains a difficult challenge, despite the need for this information to develop sound regulatory and mitigation strategies. We present results from a regional-scale study of nitrogen isotopes (delta15N) in wet nitrate deposition across 33 sites in the midwestern and northeastern U.S. We demonstrate that spatial variations in delta15N are strongly correlated with NO(x) emissions from surrounding stationary sources and additionally that delta15N is more strongly correlated with surrounding stationary source NO(x) emissions than pH, SO4(2-), or NO3- concentrations. Although emission inventories indicate that vehicle emissions are the dominant NO(x) source in the eastern U.S., our results suggest that wet NO3- deposition at sites in this study is strongly associated with NO(x) emissions from stationary sources. This suggests that large areas of the landscape potentially receive atmospheric NO(y) deposition inputs in excess of what one would infer from existing monitoring data alone. Moreover, we determined that spatial patterns in delta15N values are a robust indicator of stationary NO(x) contributions to wet NO3- deposition and hence a valuable complement to existing tools for assessing relationships between NO3- deposition, regional emission inventories, and for evaluating progress toward NO(x) reduction goals.

Habituation to auditory distractors in a cross-modal, color-word interference task.

The speed of naming the color of a colored square was examined with acoustic distraction to study the effects of the formation of a mental representation (neural model) of distractors. In Experiment 1, practice with the distractors (color words, noncolor words, and tones) was examined, and in Experiments 2 and 3 each color-naming trial was preceded by preexposures to sounds that could be dissimilar, similar, or identical to the upcoming auditory distractor. Consistency in the identity of ignored sounds, whether during color-naming practice or between preexposures and test, reduced interference with color naming. Consistency in voice played no role, and attended preexposures were ineffective in reducing interference. Given these results, the authors propose that mental representations of distractors include information about their task relevance, which modulates disruption of the primary task.

Is there a temporal basis of the word length effect? A response to Service (1998)

Service (1998) carried out a study of the word length effect with Finnish pseudowords in which short and long pseudowords were identical except for the inclusion of certain phonemes differing only in pronunciation length, a manipulation that is impossible in English. She obtained an effect of phonemic complexity but little or no word duration effect per se--a discrepancy from the expectations generated by the well-known working memory model of Baddeley (1986). In the present study using English words, we controlled for phonemic complexity differences by using the same words for the short- and long-word sets, but with instructions inducing shorter or longer pronunciation of the words. We obtained substantial word duration effects. Concerns raised by Service are addressed, and we conclude that both duration and complexity are likely to contribute to the word length effect in serial recall.

Persistence of memory for ignored lists of digits: areas of developmental constancy and change.

Contrary to the common belief that sensory memory remains unchanged across development in childhood, there have been several previous reports suggesting that the persistence of sensory memory, at least for sounds, increases with age in childhood. Because those previous studies all used isolated sounds as stimuli, it is not yet clear how this developmental difference influences the recall of sound series. The present study adapts the procedure of J. S. Saults and N. Cowan (1996), who studied memory for attended and ignored spoken words, to examine here the recall of attended and ignored lists of digit. A developmental increase in the persistence of memory was obtained only for the final item in an ignored list, which is the item for which sensory memory is thought to be the most vivid at short retention intervals.

The role of attention in the development of short-term memory: age differences in the verbal span of apprehension.

In previous studies of memory span, participants have attended to the stimuli while they were presented, and therefore have had the opportunity to use a variety of mnemonic strategies. In the main portion of the present study, participants (first- and fourth-grade children, and adults; 24 per age group) carried out a visual task while hearing lists of spoken digits and received a post-list digit recall cue only occasionally, for some lists. Under these conditions, list information presumably must be extracted from a passively held store such as auditory sensory memory. The results suggest that each individual has a core memory capacity limit that can be observed clearly in circumstances in which it cannot be supplemented by mnemonic strategies, and that the capacity limit appears to increase with age during childhood. Other, attention-demanding processes also contribute to memory for attended lists.

The nature of cross-modal color-word interference effects.

Cowan and Barron (1987) and Cowan (1989b) reported that color-naming performance was slowed by spoken color names drawn from the same set but presented in an order unrelated to the printed colors. Although Miles, Madden, and Jones (1989) and Miles and Jones (1989) were unable to replicate this cross-modal effect, it is replicated here in two experiments with much better experimental control than before. However, the effect is shown to depend upon the relative timing of the color and word in a way that conflicts with the theoretical account that Cowan and Barron offered. While Cowan and Barron suggested that an irrelevant color word would contaminate the response set if this word occupied short-term memory when the color was about to be named, it appears that interference actually occurs only if the memory representation was formed very recently and had not been inhibited. Further implications for processing are discussed.

Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase.

Mitogen-activated protein/ERK kinase kinases (MEKKs) phosphorylate and activate protein kinases which in turn phosphorylate and activate the p42/44 mitogen-activated protein kinase (MAPK), c-Jun/stress-activated protein kinases (JNKs), and p38/Hog1 kinase. We have isolated the cDNAs for two novel mammalian MEKKs (MEKK 2 and 3). MEKK 2 and 3 encode proteins of 69.7 and 71 kDa, respectively. The kinase domains encoded in the COOH-terminal moiety are 94% conserved; the NH2-terminal moieties are approximately 65% homologous, suggesting this region may encode sequences conferring differential regulation of the two kinases. Expression of MEKK 2 or 3 in HEK293 cells results in activation of p42/44MAPK and JNK but not of p38/Hog1 kinase. Immunoprecipitated MEKK 2 phosphorylated the MAP kinase kinases, MEK 1, and JNK kinase. Titration of MEKK 2 and 3 expression in transfection assays indicated that MEKK 2 preferentially activated JNK while MEKK 3 preferentially activated p42/44MAPK. These findings define a family of MEKK proteins capable of regulating sequential protein kinase pathways involving MAPK members.

Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels.

A site-directed anti-peptide antibody (anti-CNA1) directed against the alpha 1 subunit of class A calcium channels (alpha 1A) recognized a protein of approximately 190-200 kDa in immunoblot and immunoprecipitation analyses of rat brain glycoproteins. Calcium channels recognized by anti-CNA1 were distributed throughout the brain with a high concentration in the cerebellum. Calcium channels having alpha 1A subunits were concentrated in presynaptic terminals making synapses on cell bodies and on dendritic shafts and spines of many classes of neurons and were especially prominent in the synapses of the parallel fibers of cerebellar granule cells on Purkinje neurons where their localization in presynaptic terminals was confirmed by double labeling with the synaptic membrane protein syntaxin or the microinjected postsynaptic marker Neurobiotin. They were present in lower density in the surface membrane of dendrites of most major classes of neurons. There was substantial labeling of Purkinje cell bodies, but less intense staining of the cell bodies of hippocampal pyramidal neurons, layer V pyramidal neurons in the dorsal cortex, and most other classes of neurons in the forebrain and cerebellum. Scattered cell bodies elsewhere in the brain were labeled at low levels. These results define a unique pattern of localization of class A calcium channels in the cell bodies, dendrites, and presynaptic terminals of most central neurons. Compared to class B N-type calcium channels, class A calcium channels are concentrated in a larger number of presynaptic nerve terminals implying a more prominent role in neurotransmitter release at many central synapses.

Role of calcium channel subtypes in calcium transients in hippocampal CA3 neurons.

Multiple subtypes of voltage-gated calcium channels are differentially localized in brain neurons suggesting that they serve distinct roles in neuronal excitation and signaling. In organotypic hippocampal slice cultures, class D (L-type) calcium channels are predominantly located in the cell bodies of CA3 neurons while class B (N-type) and class A (P or Q-type) are localized in dendrites and associated presynaptic terminals with relatively low somal expression. Using specific antagonists to inhibit calcium transients recorded in CA3 neuronal cell bodies, we found that L-type calcium channels have a predominant role in somal calcium transients elicited by trains of strong stimuli applied to either the soma or the distal apical dendrite while class A calcium channels make a smaller contribution. Presynaptic class B (N-type) and class A (P- and/or Q-type) calcium channels are critical for glutamate-mediated synaptic transmission onto the dendrites of CA3 neurons. Postsynaptic class A and B calcium channels detected on the dendritic shaft by immunocytochemistry were not found to contribute substantially to somal calcium transients during repetitive stimulation of distal dendrites, but sodium channels were required for calcium transients elicited by somatic or dendritic stimulation. Our results show that the different calcium channel subtypes serve distinct roles in cellular activation and transmission of signals in CA3 neurons, consistent with their differential subcellular localization.

Corticosterone exacerbates kainate-induced alterations in hippocampal tau immunoreactivity and spectrin proteolysis in vivo.

Aberrant elevations in intracellular calcium levels, promoted by the excitatory amino acid glutamate, may be a final common mediator of the neuronal damage that occurs in hypoxic-ischemic and seizure disorders. Glutamate and altered neuronal calcium homeostasis have also been proposed to play roles in more chronic neurodegenerative disorders, including Alzheimer's disease. Any extrinsic factors that may augment calcium levels during such disorders may significantly exacerbate the resulting damage. Glucocorticoids (GCs), the adrenal steroid hormones released during stress, may represent one such extrinsic factor. GCs can exacerbate hippocampal damage induced by excitotoxic seizures and hypoxia-ischemia, and we have observed recently that GCs elevate intracellular calcium levels in hippocampal neurons. We now report that the excitotoxin kainic acid (KA) can elicit antigenic changes in the microtubule-associated protein tau similar to those seen in the neurofibrillary tangles of Alzheimer's disease. KA induced a transient increase in the immunoreactivity of hippocampal CA3 neurons towards antibodies that recognize aberrant forms of tau (5E2 and Alz-50). The tau immunoreactivity appeared within 3 h of KA injection, preceded extensive neuronal damage, and subsequently disappeared as neurons degenerated. KA also caused spectrin breakdown, indicating the involvement of calcium-dependent proteases. Physiological concentrations of corticosterone (the species-typical GC of rats) enhanced the neuronal damage induced by KA and, critically, enhanced the intensity of tau immunoreactivity and spectrin breakdown. Moreover, the GC enhancement of spectrin proteolysis was prevented by energy supplementation, supporting the hypothesis that GC disruption of calcium homeostasis in the hippocampus is energetic in nature. Taken together, these findings demonstrate that neurofibrillary tangle-like alterations in tau, and spectrin breakdown, can be induced by excitatory amino acids and exacerbated by GCs in vivo.

Corticosterone impairs hippocampal neuronal calcium regulation--possible mediating mechanisms.

Corticosterone (CORT), the predominant glucocorticoid of rats which is secreted during stress, increases hippocampal neuronal vulnerability to excitotoxins, hypoxia-ischemia, and hypoglycemia in an energy-dependent manner. A mechanism for this endangerment could be the CORT-induced impairment of hippocampal neuronal calcium regulation. We have shown that CORT causes an energy-dependent prolonged elevation of cytosolic free calcium ([Ca2+]i) in response to kainic acid stimulation in cultured hippocampal neurons. That study utilized the calcium-sensitive dye fluo-3, which is unsuitable for determination of basal [Ca2+]i. The present study circumvents that limitation by using the dye fura-2 AM. We have replicated the previous demonstration that CORT potentiates the [Ca2+]i response to KA; we have also observed that CORT elevates basal [Ca2+]i concentrations. Furthermore, we have observed that the mechanism for this CORT impairment of calcium regulation involves a reduction in stimulus-induced calcium efflux. Energy-dependent disruptions in neuronal calcium regulation, such as induced by CORT, have been associated with subsequent neurotoxicity. Thus, the CORT-induced impairment of hippocampal neuronal calcium regulation could be the mechanism for the neuronal vulnerability and toxicity evident following CORT treatment and stress.

Corticosterone enhances kainic acid-induced calcium elevation in cultured hippocampal neurons.

Corticosterone, a steroid secreted during stress, increases hippocampal neuronal vulnerability to excitotoxins, hypoxia-ischemia, and antimetabolites. Energy supplementation and N-methyl-D-aspartate receptor antagonists prevent this corticosterone-enhanced neurotoxicity. Because neuronal calcium regulation is energy dependent and a large calcium influx accompanies N-methyl-D-aspartate receptor activation, we investigated whether corticosterone exacerbates the elevation of hippocampal neuronal calcium induced by the glutamatergic excitotoxin kainic acid. Corticosterone caused a 23-fold increase in the magnitude of the calcium response to kainic acid, a sevenfold increase in the peak magnitude of the calcium response, and a twofold increase in calcium recovery time. This corticosterone effect may be energetic in nature as corticosterone decreases hippocampal neuronal glucose transport. Glucose supplementation reduced the corticosterone effect on the magnitude and peak magnitude of the calcium response to kainic acid. Glucose reduction, by the approximate magnitude by which corticosterone inhibits glucose transport, mimicked the corticosterone effect on the peak magnitude of the calcium response to kainic acid. Thus, corticosterone increases calcium after kainic acid exposure in hippocampal neurons in an energy-dependent manner. Elevated calcium is strongly implicated in stimulating neurotoxic cascades during other energetic insults and may be the mechanism for the corticosterone-induced hippocampal neuronal vulnerability and toxicity.

Glucocorticoids exacerbate kainic acid-induced extracellular accumulation of excitatory amino acids in the rat hippocampus.

Glucocorticoids (GCs) compromise the ability of hippocampal neurons to survive various insults, and do so, at least in part, by exacerbating steps in the glutamate/N-methyl-D-aspartate (NMDA)/calcium cascade of damage. As evidence, GCs impair uptake of glutamate by hippocampal astrocytes, the GC endangerment of the hippocampus is NMDA receptor dependent, and GCs exacerbate kainic acid (KA)-induced calcium mobilization. These observations predict that GCs should also exacerbate KA-induced accumulation of extracellular glutamate and aspartate. To test this, adrenalectomized rats were given replacement GCs in either the low or high physiological range. Three days later, rats were anesthetized and 1 mM KA was infused through a dialysis probe placed in the dorsal hippocampus. Extracellular amino acid concentrations in the dialysate were then assessed by HPLC. After KA infusion, high-GC rats (30 +/- 3 micrograms/dl) had significantly elevated concentrations of glutamate and aspartate compared with low-GC rats (all less than 0.95 micrograms/dl). The glutamate accumulation was due to GCs raising pre-KA concentrations, whereas the aspartate accumulation was due to GCs exacerbating the KA-induced rise. Glutamine concentrations were unaffected by KA, whereas the high-GC regimen elevated glutamine concentrations both before and after KA. Taurine concentrations rose after infusion of KA, but were unaffected by GC regime, whereas alanine concentrations were unaffected by either manipulation. Serine concentrations were unaffected by KA, but were depressed both before and after KA in high-GC rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.